Method of producing reticulated cellulose having type II crystalline cellulose

ABSTRACT

A method and media are provided for producing bacterial cellulose under agitated culture conditions resulting in sustained production over an average of 70 hours of at least 0.1 g/liter per hour are achieved. A unique reticulated cellulose product is produced using the methods and conditions claimed, and may be in the form of a sheet characterized by substantial resistance to densification and great tensile strength when produced by sheet forming means. Strains of Acetobacter that are stable under agitated culture conditions and that exhibit substantially reduced gluconic and keto-gluconic acids production are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/070,650, filedJun. 1, 1993, which is a continuation of U.S. Ser. No. 07/657,178, filedFeb. 19, 1991, now abandoned, which is a divisional of U.S. Ser. No.196,496, filed May 19, 1988, now U.S. Pat. No. 5,079,162, which is acontinuation-in-part of U.S. Ser. No. 900,086, filed Aug. 28, 1986, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 788,994,filed Oct. 18, 1985, now abandoned. All of these applications areincorporated in their entirely by reference herein.

FIELD OF THE INVENTION

The invention concerns strains of Acetobacter that are capable ofproducing cellulose in artificial culture. More specifically, theAcetobacter strains according to the invention are characterized by anability to produce large amounts of cellulose in agitated culturewithout manifesting instability leading to loss of cellulose productionin culture. Among the Acetobacter strains according to the invention arestrains additionally characterized by a substantially reduced ability toproduce gluconic acid and keto-gluconic acids. The production ofcellulose using such gluconate negative (glcA⁻) strains in artificialculture medium, is facilitated as these strains do not substantiallyacidify the medium, and thus increase cellulose concentration (totalgm/1) and volumetric productivity. Such gluconate negative Acetobacterstrains are useful in high cell concentration cultures.

The invention also concerns a bacterial cellulose product having novelproperties. In particular, the invention concerns a reticulatedcellulose product. This reticulated bacterial cellulose product ischaracterized by a microscopic structure unlike that of bacterialcellulose produced by cellulose producing microorganisms under staticculture conditions.

The invention also pertains to a method for producing the reticulatedcellulose product by culturing cellulose producing microorganisms forsustained periods of time, generally in excess of four hours, underagitated culture conditions. The sustained and efficient production ofbacterial cellulose under agitated culture conditions was unexpected.

BACKGROUND OF THE INVENTION

The production of cellulose by Acetobacter has been the subject ofintense study since at least the 1930's. In 1947, it was shown that inthe presence of glucose and oxygen, non-proliferating cells ofAcetobacter synthesize cellulose. Hestrin, S., Aschner, M. and Mager,J., Nature, 159:64 (1947). Since the observations of Hestrin et al.,Acetobacter has been grown with the production of cellulose under avariety of conditions. For example, when grown with reciprocal shakingat about 90-100 cycles per minute, cells have been incorporated into alarge gel mass. When grown under conditions in which the culture mediumis agitated with swirling motion for four hours, stellate gel bodiesform which are comprised of cellulose and cells. When grown asstanding-cultures, a pellicle forms at the air/medium interface. Thepellicle forms as a pad generally having the same surface shape and areaas the liquid surface of the vessel containing the culture. Hestrin andSchramm, Biochem. Journal, 58:345-352 (1954). Hestrin and Schrammobserved rapid cellulose production by freeze-dried preparations ofAcetobacter containing less than 10% viable cells. These experiments,however, only measured cellulose production in shaking conditions bysuch freeze dried preparations over a relatively short period of threeto four hours, and were run under citrate buffering conditions tocontrol significant pH changes caused by gluconic acid produced byAcetobacter in the presence of glucose.

Polysaccharide biosynthesis by Acetobacter has been studied by severalgroups using non-growing cultures. In some of these studies, Acetobacterstrain 1499 was grown, the cells were freed from the cellulose pellicle,resuspended in 0.01M Tris-EDTA, frozen, and then thawed as described inHestrin and Schramm, (1954). These treated cells were used forbiochemical studies under conditions that did not sustain growth of thecells, but which did preserve enzymatic activity permitting thecellulose to be synthesized by the prepared cells.

Progress in determining conditions for culturing Acetobacter forcellulose production, however, has not been the subject of widereporting. Thus, the conditions used for culturing Acetobacter asdescribed in U.K. patent application 2,131,701A, by Ring et al., whichclaims priority of U.S. patent application Ser. No. 450,324, filed Dec.16, 1982 (now issued U.S. Pat. No. 4,588,400), are those described inHestrin and Schramm (1954); i.e., an initial pH of about 6, temperaturesin a range from 15° C. to 35° C. and preferably 20° C. to 28° C.

According to De Ley et al., "Acetobacteriacea" pp. 267-278 in BergeysManual of Systematic Bacteriology, Kreig and Holt, eds., 1st ed.,William & Wilkins, Baltimore and London, 1984, the best carbon sourcesfor growth in descending order are ethanol, glycerol and lactic acid.Acid is formed from n-propanol, n-butanol and D-glucose. The carbonsources described in U.K. application 2,131,701A include fructose,mannitol, sorbitol and glucose, all of which give rapid celluloseproduction, and glycerol, galactose, lactose, maltose and sucrose, allof which give slower growth. No growth was observed using sorbose,mannose, cellobiose, erythritol, ethanol, and acetic acid.

In U.K. patent application 2,131,701A it is desired to produce acoherent gel-like material for use as a wound dressing, after processingto remove the culture medium. To obtain this mat-like form, theculturing material is kept motionless during cell growth and celluloseproduction for a period ranging from a few hours to days or weeks.

Although the formation of a coherent mat or pellicle in motionless orstanding culture conditions is the culture mode described in the U.K.patent application 2,131,701A, this patent further explains thatintermittent agitation of the culture medium containingcellulose-synthesizing Acetobacter can control the length of thecellulose fibril produced by the microorganism. Intermittent agitationproduces fibrils of finite length which is determined by the linearextension rate of the fibril by the microorganism and the period betweenagitative shearing of the fibril from the surface of the bacterium.Nothing, however, is disclosed about the effects of continuous agitationon the cellulose product.

The production of cellulose from Acetobacter in continuously agitatedcultures is beset with numerous problems, the most difficult of whichhas heretofore been culture instability. This instability isdemonstrated by loss of the ability to make cellulose and the gradualovergrowth of cellulose producing cells by non-producing types. Straininstability may be the result of the appearance of spontaneous mutantsor variants of the microorganism that are cellulose non-producers. Thisappearance of non-producers apparently occurs with a frequency highenough to shift the population balance of a culture fromcellulose-producing to cellulose non-producing types during growth inagitated culture. The loss of cellulose production in shaking culturesmay also be merely the result of physiological factors rather thanmutation to non-cellulose producing types due to genetic changes.Leisinger et al., Ueber cellulosefrie Mutanten von Acetobacter xylinum,Arch. Mikrobiol. 54:21-36 (1966). Although the cause is not known, thesustained production of bacterial cellulose in agitated culture mediumhas not heretofore been reported.

Cellulose negative (Cel⁻) strains of Acetobacter have been made bychemical mutagenesis with ethyl methane sulfonate (EMS), nitrous acidand N'-nitro-N-nitroso-guanidine (NG). When grown in static cultures,all of the EMS and nitrous acid-, and 90% of the NG-mutated strainsreverted to cellulose producing types. Valla et al., Cellulose-NegativeMutants of Acetobacter xylinum, J. Gen. Microbiol., 128(7):1401-1408(1982). Growth of mixed cultures of cellulose producing andnon-producing strains in static cultures strongly favored celluloseproducing strains in static cultures, whereas growth of such mixedcultures in shake flasks favored non-producing strains. Valla et al.(1982). This result lends support to the hypothesis that the cellulosemat or pellicle produced by this microorganism enables Acetobacter cellsto reach the surface of static liquid medium where the supply of oxygenis abundant. Under shaking conditions where oxygen dissolution rate andlow oxygen solubility limits growth, cellulose negative strains arefavored because of selective aggregation of cellulose producing cellsand resulting mass transfer limitation with respect to oxygen. It willthus be readily apparent that the identification and isolation ofAcetobacter strains that are stable cellulose producers in agitatedculture medium is of critical importance to large scale production ofcellulose from Acetobacter in cultures which are concentrated enough torequire agitation for sufficient oxygen supply to the medium.

Acetobacter is characteristically a gram-negative, rod shaped bacterium0.6-0.8 μm by 1.0-4 μm. It is strictly aerobic; metabolism isrespiratory, never fermentative. It is further distinguished by theability to produce multiple poly β-1,4-glucan chains, chemicallyidentical to cellulose. Multiple cellulose chains or microfibrils aresynthesized at the bacterial surface at sites external to the cellmembrane. These microfibrils have cross sectional dimensions of about1.6 nm×5.8 nm. In static or standing culture conditions the microfibrilsat the bacterial surface combine to form a fibril having cross sectionaldimensions of about 3.2 nm×133 nm.

The cellulose fibrils produced by these microorganisms, althoughchemically resembling, in many aspects, cellulose produced from woodpulp, are different in a number of respects. Chiefly among thedifferences is the cross-sectional width of these fibrils. The cellulosefibrils produced by Acetobacter are usually two orders of magnitudenarrower than the cellulose fibers typically produced by pulping birchor pine wood. The small cross sectional size of theseAcetobacter-produced fibrils, together with the concomitantly greatersurface area than conventional wood-pulp cellulose and the inherenthydrophilicity of cellulose, leads to a cellulose product havingunusually great capacity for absorbing aqueous solutions.

This capacity for high absorbency has been demonstrated to be useful inthe manufacture of dressings which may be used in the treatment of burnsor as surgical dressings to prevent exposed organs from surface dryingduring extended surgical procedures. Such uses and a variety ofmedicament impregnated pads made by treatment of Acetobacter-producedintact pellicles are disclosed in U.K. 2,131,701A. The pellicles of thisU.K. application are produced by growing Acetobacter in a culture mediumtray which remains motionless. Because Acetobacter is an obligateaerobe, i.e., it cannot grow in the absence of oxygen, production ofcellulose by Acetobacter occurs at the air-liquid medium interface. Eachbacterium continuously produces one fibril at the air-liquid interface.As new cellulose is formed at the surface, existing cellulose is forceddownward into the growth medium. As a result, cellulose pelliclesproduced in static culture conditions consist of layers of cellulosefibers. Significantly, the volume of cellulose so produced is restrictedby the interface between air and culture medium. The tendency of knownAcetobacter strain's to become cellulose non-producers when culturedunder agitated conditions at increased dissolved oxygen concentration,severely limits the amount of cellulose that can be made economically.Consequently, high cellulose productivity per unit volume of vessel inextended agitated fermentations has not been previously reported.

Another problem associated with cellulose production by Acetobacter inbatch culture, whether agitated or motionless, is the ability ofAcetobacter to convert glucose to gluconic acid and ketogluconic acids.The pH drop associated with such acid production by the organism alsolimits the amount of cellulose made, particularly in batch cultures.Moreover, the production of gluconic and keto-gluconic acids removesglucose from the medium at the expense of cellulose production.

Celluloses are encountered in various crystalline forms or "polymorphs."Celluloses have varying degrees of crystallinity depending on the sourceof the cellulose and method of treatment. Two common crystalline formsof cellulose are "cellulose I" and "cellulose II" which aredistinguishable by X-ray, Raman spectroscopy and infrared analysis aswell as by Nuclear Magnetic Resonance (NMR). Cellulose I is the latticestructure for native cellulose, and cellulose II is the latticestructure for mercerized or regenerated cellulose. Structuraldifferences between cellulose I and II contribute to differences inreactivity and many physical properties of various celluloses.

In addition to cellulose I and II, celluloses typically have someamorphous regions which are present to some extent in all native,regenerated and mercerized celluloses and which complicate structuralanalysis.

C-13 solid-state NMR has revealed the presence of two distinct forms ofcellulose I called I-alpha (I.sub.α) and I-beta (I.sub.β). These formsoccur in plant-derived celluloses as well as bacterial and algalcelluloses. The I.sub.α form dominates in plant-derived celluloseswhereas the I.sub.α form dominates in algal and bacterial celluloses(VanderHart and Atalla, Science 223: 283-284 (1984), and VanderHart andAtalla, Macromolecules 17: 1465-1472 (1984)). These forms cannot bedistinguished by X-ray diffraction but are clearly distinguishable bysolid state C-13 NMR and Raman spectroscopy.

SUMMARY OF THE INVENTION

The present invention includes a reticulated bacterial cellulose producthaving novel properties. This reticulated bacterial cellulose product ischaracterized by a microscopic structure unlike that of bacterialcellulose produced by cellulose-producing microorganisms under staticculture conditions.

The bacterial cellulose produced under known static culture conditionsis characterized by a disorganized layered structure consisting ofoverlaying and intertwisted discrete cellulose strands or fibrils. Thisdisorganized layered structure reflects the growth pattern ofcellulose-producing microorganisms which is typified by themicroorganism Acetobacter. In static cultures, Acetobacter typicallygrows at the interface of the surface of the liquid medium and air. Asthe cells grow, cellulose fibers are continuously elaborated andaccumulated, sinking deeper into the medium. The cellulose pellicle thusformed is comprised of a mass of continuous layered cellulose fiberswhich support the growing population of Acetobacter cells at the airmedium interface.

The macroscopic and microscopic structures of the cellulose produced inaccordance with the agitated culture conditions of the invention differfrom that made pursuant to the known static culture conditions.Macroscopically, the cellulose of the invention forms in the culture aspellets having diameters in the range of from approximately 0.05 mm toapproximately 10.0 mm rather than as a continuous pellicle at the airmedium interface. This pellet form remains after base extraction torecover the cellulose product and is believed to influence the physicalproperties of the final cellulose product. Microscopically (by scanningelectron microscopy (SEM)), the cellulose product according to theinstant invention is characterized by a three dimensional reticularstructure. This structure is characterized by frequently thickenedstrands of cellulose that interconnect forming a grid-like patternextending in three dimensions. The bacterial cellulose produced instatic cultures is characterized by overlapping adjacent strands ofcellulose that are oriented predominantly with the long axis of thestrand in parallel but disorganized planes. By contrast, the reticularstructure of the cellulose product according to the present invention ischaracterized by interconnecting, rather than overlapping strands ofcellulose. These interconnecting strands have both roughly perpendicularas well as roughly parallel orientations. As a result, the reticularcellulose product according to the invention has a more generallyfenestrated appearance in scanning electron micrographs, whereascellulose produced in static culture has an appearance in scanningelectron micrographs of strands piled on top of one another in acrisscrossing fashion, but substantially parallel in any given layer.The strands of the cellulose product according to the invention aregenerally thicker than those produced in comparable media withoutagitation. The reticulated cellulose was composed of interconnectingfilaments ranging in width from about 0.1 to about 0.2 microns. Thefilaments or strands of cellulose produced under non-agitated conditionsranged in width from about 0.05 to about 0.2 microns with many strandsin the range of 0.05 to 0.10 microns.

In addition, the fibrils of the non-reticulated cellulose product ascompared to the fibrils of the reticulated product appear to branch andinterconnect less frequently. Although the non-reticulated celluloseproduct appears to have many fibrils that contact one another, thefibrils overlay one another rather than interconnect. By contrast, thefibrils of the reticulated cellulose according to the invention, have alarge proportion of fibers that interconnect or intertwine to form asubstantially continuous three-dimensional network of interconnectingfibers.

The reticulated cellulose product according to the invention has severaladvantages over cellulose produced under non-agitated conditions.Because the reticulated cellulose product is characteristically producedin agitated cultures of cellulose producing microorganisms such asAcetobacter, it can be produced using conventional high volumefermentation methods. Thus, unlike the production of cellulose pelliclesin the slow growing, non-agitated culture media of the prior art, thereticulated cellulose product of the present invention may be producedin fast growing cultures of Acetobacter with high volumetricproductivity and high concentration of the reticulated celluloseproduct.

One way the reticulated cellulose product according to the invention canbe distinguished from bacterial cellulose produced under non-agitatedconditions is by its characteristics upon consolidation into apaper-like sheet. Batches of the reticulated cellulose product generallyoffer a high resistance to densification when formed into a sheet byconventional means. By use of different wet pressing loads, a series ofsheets was prepared having densities in the range of about 300 to about900 kg/m³, with those exhibiting substantial resistance to densificationbeing about 300 to about 500 kg/m³. In spite of the low densities, thesepaper like sheets have very high tensile strength as measured accordingto Technical Association of the Pulp and Paper Industry (TAPPI) methodT494 om-81 using an Instron Universal test instrument. Typically, thetensile index for sheets made from reticulated cellulose of the densityrange of 300-500 kg/m³ is between 100 and 150 Newton-meters/gram.Comparable sheets formed from kraft pulp having densities below about500 kg/m³ have virtually no tensile strength.

Handsheets formed from cellulose produced under static cultureconditions do not exhibit the above-mentioned resistance todensification. Typically, such sheets from non-agitated cultures ofcellulose have densities from about 500 to about 750 kg/m³ depending onthe wet pressing load employed.

The invention also pertains to a method for producing the reticulatedcellulose product by culturing cellulose producing microorganisms forsustained periods of time under agitated culture conditions. Theproduction of bacterial cellulose under agitated culture conditions issurprising in light of the well known tendency of agitated cultureconditions to select for cellulose non-producing strains of Acetobacter.Valla et al., (1982). Moreover, the reticulated structure of thecellulose produced under these conditions is entirely unexpected.

As used herein, the term Acetobacter refers to a genus ofmicroorganisms, and in particular, to the members of that genus thatproduce cellulose. Although a number of microorganisms fitting thisdescription are known, their taxonomic classification has been subjectto debate. For example, the cellulose producing microorganisms listed inthe 15th Edition of the catalogue of the American Type CultureCollection under accession numbers 10245, 10821, and 23769 areclassified both as Acetobacter aceti subsp. xylinum and as Acetobacterpasteurianus. Thus, any cellulose producing strain of Acetobacterwhether classified as Acetobacter aceti subsp. xylinum, Acetobacterpasteurianus or otherwise, that has the characteristics of stabilityunder agitated culture conditions as further explained below, isconsidered to be within the scope of the invention.

The inventors have discovered and developed a number of strains ofAcetobacter that are stable in long term cultures under bothnon-agitated and agitated culture conditions including fermentor processconditions. The stability of the strains is demonstrated under agitatedconditions; the strains according to the invention generally appear tochange to cellulose non-producing types at a very low frequency, on theorder of less than 0.5% at the end of a fermentation run of 42-45generations (including the seed and pre-seed stages), as determined bycolony morphology when subcultures of Acetobacter grown in liquid mediumunder agitated conditions are plated on solid medium.

The Acetobacter strains according to the invention have been mutagenizedand a number of derivative strains have been selected. At least two ofthe selected strains are characterized by a sharply reduced ability toform gluconic and keto-gluconic acids when grown on a glucose containingmedium. Such strains having a reduced ability to form gluconic acid arestable. At the end of a fermentation run of 42-45 generations (includingthe seed and pre-seed stages), less than 0.5% gluconic and ketogluconicacid producing types are detected as determined by the inability ofcells from the fermentor broth to form calcium carbonate-clearingcolonies on agar plates containing glucose. These strains are stablewith respect to change to cellulose non-producing type and to change togluconic and keto-gluconic acids producing type.

Various feed stocks may be used as the carbon source for growth of thecellulose-producing microorganisms and reticulated cellulose productaccording to the invention so long as they are supplied free ofcontaminating organisms. Appropriate carbon sources include themonosaccharides and disaccharides in pure or partially purified form oras monosaccharide and disaccharide-containing stocks such as hydrolyzedcorn starch, hydrolyzed wood and molasses.

Cellulose production with the strains according to the invention may becarried out under conditions permitting higher dissolved oxygenconcentration than possible under standing conditions. The ability ofthe strain to maintain cellulose production while agitated permitsvarious means for increasing dissolved oxygen in the culture medium tobe used. Thus, direct agitation of the culture medium by impellersimmersed in the medium has been used successfully, although adherence ofthe cellulose produced to the impeller blades can be a disadvantage forsmall-scale production. Means for agitating the culture which increasedissolved oxygen content are well known to those familiar with microbialfermentation. Oxygen tension in the broth can vary between 0.01 to 1.0atmosphere oxygen.

In tests in a fermenter (14 liters) using an impeller to agitate thebroth, it was found that the characteristics of the broth (viscosity)and the resulting cellulose (particle size and morphology, settlingrate, hand sheet formation) are affected by high impeller speeds (aboveabout 600 rpm in the runs carried out). These effects were morepronounced the longer the cultures were agitated at such speeds. It isnot known whether these results will apply to all fermenter volumes andconfigurations and/or methods of agitation. In the tests conducted,however, the higher impeller speeds/longer agitation times resulted insmaller particles determined by longer particle settling times, highersuspension viscosity, less cellulose retained in handsheet tests.Accordingly, depending on the intended end use for the cellulose, it maybe desirable to avoid culturing the organisms under such extremeagitation conditions. It is, therefore, preferred to carry out thefermentation at sufficiently low agitation rates and agitation times soas to avoid any substantial degradation of the properties of thecellulose product.

The effective pH range for culturing the cellulose producingmicroorganisms according to the invention is between 4 and 6, with apreferred range of 4.5 to 5.5, and most preferably pH 5. pH may becontrolled by means of buffers such as citrate or 3,3 dimethylglutaricacid added to the culture medium; or the addition of base or; acid tothe medium in an amount sufficient to maintain pH in the desired range.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photographs showing the macroscopic structure of thereticulated cellulose product in pellet form of the present invention.FIG. 1A shows the product before base extraction; FIG. 1B shows it afterbase extraction and purification. The dividing lines are approximately 1mm apart.

FIG. 2 is a scanning electron micrograph at magnification of 5000× ofnon-reticulated cellulose produced under non-agitated conditions.

FIG. 3 is a scanning electron micrograph at a magnification of 5000× ofthe reticulated cellulose of the present invention.

FIG. 4 is a scanning electron micrograph at a magnification of 10,150×of non-reticulated cellulose produced under non-agitated conditions.

FIG. 5 is a scanning electron micrograph of a magnification of 10,330×of reticulated cellulose.

FIG. 6 is a graph showing growth (increase in biomass) of Acetobacterstrain 1306-21 in various media as described in Example XVI, infra.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description of the invention a number ofculture media are mentioned. Unless otherwise indicated the media areformulated as is indicated below.

R20-2 medium has the following composition:

    ______________________________________    R20-2    ______________________________________    Bacto-peptone      5 g/l    Yeast Extract      5 g/l    Na.sub.2 HPO.sub.4 5 g/l    Citric Acid        1.15 g/l    Carbon Source      As specified (if not                       specified, 2% glucose)    Final pH           5.0 +/- 0.2    ______________________________________

R20 is the same as above but the final pH is 6.0. R20-3 is the same asabove but citric acid is omitted.

Y-1 medium, also referred to as minimal medium R70 or AcetobacterMinimal Medium (AMM), has the following composition:

    ______________________________________    Compound    Final Concentration (mM)    ______________________________________    (NH.sub.4).sub.2 SO.sub.4                25    KH.sub.2 PO.sub.4                7.3    MgSO.sub.4  1.0    FeSO.sub.4  0.013    CaCl.sub.2  0.10    Na.sub.2 MoO.sub.4                0.001    ZnSO.sub.4  0.006    MnSO.sub.4  0.006    CuSO.sub.4  0.0002                pH = 5.0    Glucose     2% or 4% (w/v)                unless otherwise                specified    ______________________________________

For all studies using Y-1 medium the following vitamin mix was added tothe minimal medium at a 100 fold dilution:

    ______________________________________    Vitamin Mix    Compound           mg/L    ______________________________________    Inositol           200    Niacin             40    Pyridoxine HCl     40    Thiamine HCl       40    Ca Pantothenate    40    Riboflavin         20    Para-aminobenzoic acid                       20    Folic Acid         0.2    Biotin             0.2    ______________________________________

Corn steep liquor (CSL) medium has the following composition:

    ______________________________________    Ingredient    Final Concentration (mM)    ______________________________________    (NH.sub.4).sub.2 SO.sub.4                  25    KH.sub.2 PO.sub.4                  7.3    MgSO.sub.4    1.0    FeSO.sub.4    0.013    CaCl.sub.2    0.10    Na.sub.2 MoO.sub.4                  0.001    ZnSO.sub.4    0.006    MnSO.sub.4    0.006    CuSO.sub.4    0.0002    vitamin mix (above)                  10 ml/liter    carbon source as specified (usually                  glucose 2 or                  4%, w/v)    corn steep liquor                  as specified (usually    (supernatant fraction                  2 or 5%, v/v)    after centrifugation)    Antifoam      0.01% (v/v)    final pH = 5.0 ± 0.2    ______________________________________

The composition of corn steep liquor varies depending on supplier andmode of treatment. A typical corn steep liquor sample, Type E804obtained from Corn Products Unit, CPC North America, Stockton, Calif. isdescribed below:

    ______________________________________    Major Components    Percent    ______________________________________    Solids              43.8    Crude protein       18.4    Fat                 0.5    Crude fiber         0.1    Ash                 6.9    Calcium             0.02    Phosphorous         1.3    Nitrogen free extract                        17.8    Non-protein nitrogen                        1.4    NaCl                0.5    Potassium           1.8    Reducing sugars (as dextrose)                        2.9    Starch              1.6    pH                  4.5    ______________________________________

Y3-3 medium has the following composition:

    ______________________________________    Component     Concentration    ______________________________________    Yeast extract 10           g/l    Peptone       10           g/l    KH.sub.2 PO.sub.4                  4            mM    K.sub.2 HPO.sub.4                  6            mM    Glucose       20           g/l    pH            6.0    ______________________________________

R70-2 medium is a modified form of AMM. R70-2 has the followingcomposition:

    ______________________________________    Ingredient      Final Concentration (mM)    ______________________________________    (NH.sub.4).sub.2 SO.sub.4                    25    KH.sub.2 PO.sub.4                    7.3    Na Citrate      4.0    MgSO.sub.4      1.0    FeCl.sub.3      0.01    CaCl.sub.2      0.10    Na.sub.2 MoO.sub.4                    0.001    ZnSO.sub.4      0.005    MnSO.sub.4      0.005    CuSO.sub.4      0.001    CoCl.sub.2      0.001    NiCl.sub.2      0.001    vitamin mix (see below)                    10 ml/liter    Glucose         as specified                    (usually 2 or                    4%, w/v)    final pH = 5.0 ± 0.2    ______________________________________    Vitamin Mix    Compound        mg/l in mix    ______________________________________    Inositol        200    Niacin          40    Pyridoxine HCl  40    Thiamine HCl    40    Ca Pantothenate 40    Riboflavin      20    PABA            20    Folic Acid      0.2    Biotin          0.2    ______________________________________

One aspect of the invention concerns a number of stable celluloseproducing strains of Acetobacter. The stability of Acetobacter strainsaccording to the invention is demonstrated by a very low frequency ofconversion to phenotypes that do not produce cellulose. The frequency ofconversion to phenotypes that do not produce cellulose is less than5×10⁻³ as determined by colony morphology when subcultures ofAcetobacter grown under agitated conditions are plated on solid mediumat the end of a fermentation cycle of 42-45 generations. The colonies ofthe cellulose-producing strains on solid medium are generally beige orwhite and are small, raised or convex and compact in size. By contrastcellulose nonproducing strains form large, usually flat colonies onsolid medium. In addition to Acetobacter other cellulose producingbacteria such as organisms in the genus Agrobacterium may be fermentedin agitated culture to produce cellulose as described herein.

The stable Acetobacter strains according to the invention were derivedfrom an initial isolate of an A. xylinum strain obtained from theNorthern Regional Research Laboratory, Peoria, Ill. USA under AccessionNo. NRRL B42. Growth of the NRRL strain on agar plates of R20-2 mediumrevealed two colony morphologies, one white, the other beige.Microscopically, the beige colonies have the elongated rod shape cellstypical of the Acetobacter strain. This strain is designated 1306-3.Unlike the parent NRRL B42 strain, 1306-3 produces no water solublepolysaccharide as reported by Couso, R. O. et al., Biosynthesis ofPolysaccharides in Acetobacter xylinum; Sequential Synthesis of aHeptasaccharide Diphosphate Prenol; Eur. J. Biochem. 123:617-627 (1982).Cultures of 1306-3 are stable in both microscopic morphology andmacroscopic colony morphology when subcultured on different mediacontaining a variety of carbon sources. Furthermore, colony and cellularmorphology of the strain according to the invention remain stablewhether grown in static or shaking liquid cultures in various media.

Strain 1306-3 and its progeny are capable of producing cellulose in avariety of liquid culture media having various carbon and nitrogensources. Casein hydrolysate, protein hydrolysate, yeast extract, maltextract, ammonium salts, corn steep liquor and other nitrogen-richsubstances can be used as a general source of amino acids, nitrogen,minerals and vitamins. Corn steep liquor is preferred in a range between0.1% and 10% (v/v). 3% (v/v) corn steep liquor is preferred for shakingflask cultures. In fermentors an initial concentration of 2% (v/v) cornsteep liquor is supplemented during the fermentation run with anadditional 2% (v/v) corn steep liquor. Numerous carbon sources may beused including mannitol, sorbitol, sucrose, fructose and glucose,although using the latter carbon source, D-gluconic acid andketogluconic acids, including 2-keto-D-gluconic acid or5-keto-D-gluconic acid, are produced by strain 1306-3.

Acetobacter strains according to the invention that producesignificantly lower amounts of D-gluconic acid have also been developedby the inventors and are further described herein below.

Carbon sources useful in the production of the reticulated celluloseproduct may be characterized as monosaccharides or mixtures thereof suchas glucose and fructose, disaccharides such as sucrose, and mixtures ofmonosaccharide and disaccharides. In addition, the carbon source can besupplied as a complex mixture of sugars such as molasses, or plantbiomass hydrolysates such as wood hydrolysate, straw hydrolysate, cornstalk hydrolysate, sorghum hydrolysate and the like.

The concentration of monosaccharides and disaccharides or mixturesthereof may vary. Glucose alone and fructose alone in a range of 0.5 to7% (w/v) with a preferred concentration of about 4% (w/v) have beenused. In addition, mixtures of glucose and sucrose in a ratio from 1:10to 10:1 (w/w) having a total of 0.5 to 7% (w/v) of the medium may beused. A concentration of 1% (w/v) glucose and 2% (w/v) sucrose ispreferred in flask cultures. In fed batch fermentation in which thecarbon source is intermittently or continuously supplied duringfermentation, the total carbon substrate added can vary between 4 to 30%(w/v). The carbon source may be supplied as a purified or partiallypurified feed stock or alternatively as a complex mixture such asmolasses. Such carbon sources are pretreated so that they are free ofcontaminating organisms.

The conversion of glucose to D-gluconic acid on glucose containingmedium leads to a significant drop in pH of the medium in batch culture.Since pH below about 4.0 may limit growth of the cells, pH control isdesirable. pH control in liquid media culture of gluconic acid producingstrains according to the invention can be effected by use of bufferssuch as citrate. However, the amount of buffer that can be added toneutralize the acid is limited and growth of gluconate-producing strainsto high density is limited by the amount of buffer which can be added.In addition, the use of citrate or other salts as buffers adds to theexpense of the culture medium. pH control can also be effected by usingfructose as the carbon source, since Acetobacter does not metabolizefructose to acid. However, fructose is an expensive substrate andincreases the production cost of the cellulose fiber.

By treating strain 1306-3 with a mutagen, the inventors have developedstable variant strains exemplified by strains 1306-11 and 1306-21. Thesestrains produce significantly reduced amounts of gluconic acid, andketo-gluconic acids yet produce cellulose in a stable manner typical ofthe parent strain 1306-3. Mutagenesis was accomplished using aconcentration of ethyl methane sulfonate (EMS) sufficient to yield asurvival rate of approximately 1%. In the case of strain 1306-11 of thesurviving mutagenized bacteria, 8100 colonies were screened. Twoisolates produced reduced amounts of gluconic acid and keto-gluconicacids.

Strain 1306-11 was selected by culturing on plates of R20-CaCO₃ mediumand screening for colonies having a morphology similar to the parentstrain 1306-3 but which do not clear calcium carbonate in the medium.Other pH sensitive assays capable of detecting the absence of pHreducing substances such as gluconate can also be used.

Strain 1306-21 was selected as described in Example V, infra.

Bacterial cultures may be grown under agitated culture conditions by anymeans known to generate turbulence in the liquid culture medium. Suchmeans are well known to those skilled in the fermentation arts. At smallscale, generally less than 1 liter culture volume, liquid cultures maybe agitated by reciprocal or shaking incubators, which impart a swirlingmotion to the medium.

Various reactor designs may be appropriate for the large scaleproduction of the cellulose product according to the invention. See,e.g., Chapter 7 of Biochemical Engineering and Biotechnology Handbook,Atkinson and Mavituna, eds., 1st Ed. The Nature Press, New York, 1983.For large scale cellulose production, generally culture volumesexceeding 1 liter, the culture broth may be agitated by a number ofmeans including various types of impellers, buoyant lift fermentorsincluding air lift fermentors, pump driven recirculation of thefermentor broth or combinations thereof. Desirable characteristics forthe fermentor impellers include high mass transfer rates, highcirculation rates and low shear stress.

So long as the culture medium is agitated, various fermentation methodsare appropriate for growing the cellulose producing microorganism at anaverage volumetric productivity for cellulose, which is within the scopeof the invention, of at least 0.1 g/l/hr for sustained periods of time.Appropriate fermentation methods include batch fermentation, fed batchfermentations, repeated batch fermentation, and continuous fermentation.In batch fermentations the cellulose-producing microorganism isinoculated into a fermentation vessel and the fermentation proceedswithout further addition of culture media. At the end of thefermentation, the contents of the fermentation vessel are collected andthe cellulose is removed. In fed batch fermentations, various nutrients,such as carbon source or nitrogen source are added to the medium duringthe fermentation run without removing the fermentation broth forprocessing until the end of the fermentation run. The nutrients may beadded continuously, at predetermined intervals or when nutrient levelsin the medium fall below desired values. In repeated batchfermentations, a volume of the culture broth is removed for processing,a volume of fresh medium is added to the culture broth remaining in theculture vessel, and the fermentation is resumed. In repeated batchfermentations, as in fed batch fermentations, the nutrients may be addedcontinuously, at predetermined intervals or when nutrient levels in themedium fall below desired values. In continuous fermentations, the brothis removed from the fermentation vessel and replaced with fresh mediumat a constant rate. In continuous fermentations, by adjusting the flowof medium into and culture broth out of the vessel, the specific growthrate of the cellulose producing microorganism can be maintained at anapproximately constant rate.

Batch fermentations, fed batch fermentations, repeated batchfermentations, and continuous fermentations are all suitable forachieving an average volumetric productivity of at least 0.1 g/l/hr. solong as the inoculum of the culture medium is initially at least 1%(v/v). An inoculum in a range of 1-10% (v/v) of the culture medium iseffective to obtain an average volumetric productivity of cellulose of0.1 g/l/hr. An inoculum of about 5-10% (v/v) of the culture medium ispreferred. In continuous cultures, as medium, cellulose-producing cellsand cellulose are removed, fresh medium will be added at a ratesufficient to maintain the volumetric productivity at an average of atleast 0.1 g/l/hr.

To determine cellulose concentration and volumetric productivity,cellulose produced by any of the above mentioned fermentation methods isharvested from the fermentation broth. In general, any method forseparating the cellulose may be used, but centrifugation is preferred.Each batch of fermentation broth containing the cellulose-producingmicroorganism, used medium, and cellulose, is centrifuged. The volume ofthe supernatant medium is determined and the supernatant is discarded.The pellet comprising solid matter including the micro-organisms andcellulose is retained. The pellet is washed several times with deionizedwater to remove residual medium. FIG. 1A shows the macrostructure of thepellet at this stage. The retained matter is treated with an alkalisolution such as 0.1M NaOH or KOH at 60°-65° C. for at least two hours.The suspension may be mixed to disperse large clumps of cellulose.During the alkali treatment, the mixture is slowly stirred andmaintained at 60°-65° C. The alkali treated material is centrifuged andthe pellet is then washed and centrifuged three or four times indeionized water. FIG. 1B shows the macrostructure of the cellulose atthis stage. The cellulose is then dried in a vacuum oven and is weighed.Volumetric productivity is defined as total mass of cellulose producedper volume of medium used per fermentation time (g/l/hr) frominoculation to harvest for batch cultures.

The form of the cellulose, such as that shown in FIGS. 1A and 1B isdependent on the growth and hydrodynamic conditions in the fermentorused, and in the case of FIG. 1B also depends on the hydrodynamic andother physical conditions during purification and recovery of thecellulose. The important hydrodynamic factors affecting the form andquality of the cellulose product, such as effects on pellet morphologyand size and the ability to make handsheets, include shear stresses;normal stresses; turbulent eddy stresses; and pressure and cavitationforces during fermentation, purification and recovery. These factors inturn are dependent on the size and configuration of the vessels used;the type of impellers used; the agitation rates; the gas sparge rates;the temperature of the cellulose broth; the pH of the cellulose broth;the pressure to which the cellulose broth is subjected; the rheology ofthe cellulose broth; composition and total mass of the cellulose broth;the pumps and other means of transporting the cellulose broth used; andthe time under the above conditions.

In addition to scanning electron microscopy, the structure of thereticulated cellulose of the invention produced in agitated culture maybe investigated and compared to that of cellulose produced in staticculture using C-13 NMR spectra. In order to analyze the various forms ofcellulose, it was necessary to develop suitable NMR reference spectrafor three crystalline forms of cellulose, I.sub.α, I.sub.β and II. Aspectrum for cellulose II was generated from a sample of highlycrystalline cellulose II. Whatman CF-1 filter paper is derived fromcotton and is known to have a high I.sub.β content, whereas bacterialcellulose has a high I.sub.α content. The reference spectra for I.sub.αand I.sub.β were generated by the procedure described by VanderHart etal., Macromolecules, supra, incorporated by reference herein, bysubtracting an appropriately scaled spectrum obtained by NMR analysis ofWhatman CF-1 hydrocellulose powder from the spectrum obtained forcellulose sample A-012-6 (from non-agitated culture, Acetobacter strain1306-8) to generate the cellulose I.sub.α spectrum, and subtracting afraction of a scaled A-012-6 spectrum from the CF-1 spectrum to generatethe I.sub.β spectrum. This approach successively eliminates the I.sub.βand I.sub.α components. These reference spectra may then be used todetermine the I.sub.α, I.sub.β and cellulose II components of eachbacterial cellulose sample (agitated-and static) by obtaining the NMRspectrum for a sample of bacterial cellulose, then successivelysubtracting the reference spectra. The remaining NMR signal and itstotal intensity represents largely the amorphous cellulose aftercorrecting for the residual signal at 90 PPM. The amorphous cellulosecontent is then independently verifiable by analysis of the C-4resonance of the original cellulose spectrum which has distinguishablecrystalline and amorphous components. In addition, there is a residualcomponent peak that is not accounted for by the other four componentsand represents a minor impurity present in nearly all of the samples.

Using this C-13 NMR methodology, the structures of samples of bacterialcellulose produced under agitated conditions were compared to thestructure of samples produced under static culture as described below inExample XIX. Reticulated bacterial cellulose from agitated culturesexhibited a microstructure significantly different from that ofcellulose produced in static culture.

The following examples are merely to be exemplary of the invention andare not intended to be limiting.

EXAMPLE I

Cellulose Production in Static Culture

This example shows cellulose production by A. xylinum 1306-3 growing onfructose as the main substrate under non-shaking conditions. In thisexample (and examples II and XI) fructose was used instead of glucose toavoid acidification of the media by acid producing Acetobacter strainsfrom oxidation of glucose to gluconic acid and keto-gluconic acids.Strain 1499-1 obtained from Dr. Moshe Benziman, Hebrew University,Jerusalem, Israel was grown under identical conditions for purposes ofcomparison.

Seed flasks were set up containing 25 ml of Y3-3 medium with 2% fructosein 50 ml Erlenmeyer flasks and were inoculated from agar slants. Thecultures were grown for three days at 30° C. as standing cultures. Theflasks were vigorously shaken by hand to release cells and 0.5 ml ofthis culture (without cellulose pellicle) was inoculated into severalflasks of identical Y3-3 medium with 2% w/v fructose (25 ml in 50 mlflasks). Equal inoculation of all strains was accomplished using 0.03 ODat 670 nm cell suspension and inoculating 0.5 ml into each flask. Theflasks were incubated at 30° C. without shaking.

Flasks were removed from incubation and the entire contents wereharvested for sampling. Each strain was sampled in duplicate at eachtime point. Sampling was started when growth was evident and two sampleswere normally taken each day.

To measure cellulose production, the flask contents of each sample weretransferred to a 100 ml beaker. The suspension was then macerated forone minute with a large Tekmar probe at 50% of full power. Thesuspension was centrifuged at 5,000 rpm for 10 minutes. The supernatantwas discarded and the pellet was resuspended in 15 ml 1.0N NaCl salineand vortexed. The suspension was allowed to equilibrate for 15 minuteswith occasional vortexing. The sample was again centrifuged and theabove wash step repeated.

The pellet from the second wash was resuspended in 15 ml of 0.5N KOH andincubated at 60° C. with mild agitation for 120 minutes. The suspensionwas centrifuged and the KOH supernatant discarded. The pellet wasresuspended in 15 ml deionized H₂ O and left at room temperature toequilibrate for 15 minutes with occasional vortexing. The sample wascentrifuged and the above wash procedure was repeated for a total offour washes.

After the last centrifugation step the wet cellulose mat was dried at55° C. under vacuum overnight and then weighed.

                  TABLE 1    ______________________________________            Cellulose (g/l)            Time (hr)    Strain    30     60       114  138    162  234    ______________________________________    CMCC 1306-3              0.34   0.94     1.1  1.3    1.6  2.66    CMCC 1499-1              N.D.   0.19     0.51 0.67   0.72 1.30    ______________________________________     Cellulose production by strain 13063 and strain 14991 on Y33 medium with     2% fructose. The initial pH was 6.0, N.D. = Not determined

EXAMPLE II

Cellulose Production in Agitated Cultures: Stability Studies

The stability of cellulose synthesis in strains 1306-3 and 1499-1 wasexamined during serial transfers of the strains in liquid agitatedcultures, using a homogeneous (well mixed) inoculum in each transfer.Observation of cellulose production in the flasks and appearance oflarge colonies (L-colonies) from samples on plates was used to evaluatethe stability of cellulose synthesis. Cellulose production in 1499-1appeared to be unstable in aerobic agitated flasks as is shown by areduction in the amount of cellulose produced and the appearance ofincreasing numbers of large diffuse colonies which represent cellulosenon-producers. Cellulose production in 1306-3 appears to be stable forat least 30 generations in agitated flasks.

Seed cultures were inoculated from agar slants of the parental cultureinto 25 ml of R-20 medium with 2% fructose at pH 5.0 in 50 ml Erlenmeyerflasks. The seed culture was grown for four days at 30° C. as a standingculture. The flasks were shaken vigorously by hand to release cells and0.5 ml of this culture (without cellulose pellicle) was inoculated intoseveral flasks of the R20 medium with 2% fructose (25 ml in 50 mlflasks). These flasks were incubated at 30° C. without shaking.

After several transfers in standing flasks the 36 hour pellicle wasvigorously shaken and the supernatant was used as inoculum (1% v/v) tothe flasks containing R20 with 2% fructose, pH 5. The flasks (25 mlmedium in 125 ml flasks) were incubated at 30° C. and at 200 rpm in aNew Brunswick gyratory shaker. After 24 to 48 hours the culture wasaseptically blended and used as inoculum for second transfer into freshmedium and for streaking on plates. Each strain was examined for fourtransfers which is roughly equal to 30 generations. The medium used forplate experiments was R20 with 2% fructose, 1.5% agar, pH 5.0.

During growth in shake flasks the culture of strain 1499-1 appeared as afine suspension of cells and irregular clumps of different sizes. Theratio of clumps to cell suspension decreased from first transfer to thethird transfer. This observation was correlated with a significantdecrease in the amount of cellulose produced from first transfer to thethird transfer, and an increase in the fraction of L-colonies whichrepresent cellulose-non-producing strains.

The frequency of L-colonies in strain 1499-1 as a function of growth isshown in Table 2.

                  TABLE 2    ______________________________________    Instability of strain 1499-1 in    agitated culture    Stage             % L- forms    ______________________________________    Stock             8.5    Inoculum to shake flasks                      8.0    End of first shaking stage                      18.0    End of third shaking stage                      33.0    ______________________________________

In sharp contrast, strain 1306-3, during growth in shake flasks,appeared as irregular clumps of different sizes with clear mediumbetween them. Cellulose negative cells appear as single cells in thebroth and cause turbidity in the medium between the clumps. No changesin the culture appearance or in the amount of cellulose produced wereobserved after four transfers. During that time the colonies on platesappeared to be homogeneous; no large, non-producing colonies wereobserved.

EXAMPLE III

Mutagenesis of Strain 1306-3

Acetobacter strain 1306-3 was mutagenized with 1% or 2% of the chemicalmutagen ethyl methane sulfonate (EMS) and the surviving cells werescreened for loss of the ability to produce gluconate (glcA-) andketo-gluconates. Two glcA- isolates were obtained. The mutagenesis wascarried out as follows:

Two day old culture of 1306-3 on R20-2 medium was used for the mutagenictreatment. Conditions were chosen to give about 99% kill. The conditionsselected were 0.1M potassium phosphate buffer, pH 6.0; 2% EMS andincubation at 28° C. for 60 minutes. Cell concentration wasapproximately 5×10⁷ cells/ml. After this treatment the culture was keptfrozen at -80° C. for further use.

EXAMPLE IV

Screening for Gluconate Negative Mutants

Screening to obtain gluconate negative mutants of Acetobacter strain1306-3 was done with the culture obtained from the EMS mutagenesisdescribed in Example III. Screening was done on R20-2 agar platescontaining 1% CaCO₃. These plates were made by adding sterile 20% CaCO₃to sterile R20-2 to a final concentration of 1%, mixing well, anddispensing 10 ml per plate. Final pH of the plate medium was 6.0.

Plates were incubated at 30° C. and scored after 7-10 days. Coloniesthat had no zones of clearing were picked for verification screen. Thecolonies were suspended in sterile test tubes that contained 2 ml ofR20-2 medium and incubated three days to check for pellicle formationand drop in pH of the broth. Of 8100 colonies screened, two isolatesfrom the 2% EMS treated sample were found to be gluconic acid negative;the better of them was designated as 1306-11.

Production of gluconic acid by 1306-11 and 1306-3 was determined inliquid R20-2glucose and Y1-glucose. Y1 with 2% glucose had an initial pHof 4.21. The initial pH of R20-2 was 5.0.

A loopful of 1306-11 or 1306-3 culture from an R20-2 plate was suspendedin 2 ml of Y1 without carbon source. Cell counts for the suspensionswere approximately 1.6×10⁸ cells/ml. Tubes (16×125 mm) containing 3 mlof the appropriate medium were inoculated with 200 μl of the cellsuspension and mixed well by vortexing. Tubes were incubated, withoutshaking, at 30° C. for three days.

Table 3 shows the pH and gluconate levels at day three for the mutant1306-11 and for 1306-3, the parent strain. Values are shown for each ofthe duplicate tubes.

                  TABLE 3    ______________________________________    Gluconate production by Acetobacter    strains 1306-3 and 1306-11             pH               GlcA!, mM    ______________________________________    1306-3    R20-2      3.27   3.29       32.5  35.1    Y1-Glc     2.75   2.78       58.7  52.7    1306-11    R20-2      5.11   5.11       0.096 0.066    Y1-Glc     3.57   3.61       0.963 0.963    ______________________________________

EXAMPLE V

Preparation and Identification of Acetobacter strain 1306-21

Strain 1306-3 was mutagenized as described in Example III. Organismswere then plated out in CSL plates (2% glucose, 3% corn steep liquor) toestablish single colonies. Colonies were picked and placed in microtitertray wells containing 0.25 ml of CSL medium (4% glucose, 1% corn steepliquor). The trays were incubated 4-5 days until a clear drop in mediumpH as measured with pH paper (range 2.9 to 5.2) was observed. Colonieswhose pH was approximately 5 (pH paper green or greenish in color) werepassed to a secondary screen.

In the secondary screen, selected colonies were inoculated into testtubes containing 2 ml of the high glucose medium used in the microtitertray wells as described above. The tubes were incubated at 30° C.Colonies were examined for pellicle formation and pH. A gluconic acidnegative strain designated 1306-21 was selected in this manner.

For comparison purposes, samples of strains 1306-11 and 1306-21 weregrown in seed flasks containing CSL medium with varying amounts ofglucose and corn steep liquor using the general procedure described inExample XI, infra. Cellulose production and medium pH were determinedafter five days of incubation. These determinations are reported inTable 4 below.

                  TABLE 4    ______________________________________    Cellulose Production and pH of Acetobacter    Strains 1306-11 and 1306-21    pH                  Cellulose (g/L)    Medium  1306-11  1306-21    1306-11                                       1306-21    ______________________________________    CSL 2,2*            3.6      4.5        3.0    3.3    CSL 4,2 3.1      3.4        2.9    4.8    CSL 4,3 3.2      3.9        3.6    6.2    ______________________________________     *numbers indicate % glucose, % corn steep liquor, respectively.

As reported, strain 1306-21 exhibited lower acid production (higher pHs)and greater cellulose production than did strain 1306-11 in these tests.The screening method described in Examples IV and V can also be used forselection of spontaneous mutants as well as wild strains from naturewith low acid production.

EXAMPLE VI

Preparation of Reticulated Cellulose Product By Acetobacter Strain1306-14

Strain 1306-14 was obtained by EMS mutagenesis (as described above inExample III) of Acetobacter strain 1306-11. It was identified as a largewhite mucoid colony on R20-2 plates whereas 1306-11 colonies arecharacteristically even, convex and dark beige in color.

A sample of 1306-14 from a frozen stock was inoculated into 100 ml R20-2medium and was grown in static conditions at 30° C. for about threedays. The entire contents of the culture were transferred to a sterileblender. Using a small blender head, the culture was blended with shortfive second bursts. A 5% (v/v) inoculum of the disrupted culture wastransferred into 400 ml of R20-2 medium in a 2000 ml baffled flask, andwas cultured with shaking at 125 rpm at 30° C. for about 2.5 days. Thecontents of the flask were blended and were used to inoculatefermentors.

A 5% (v/v) inoculum of the disrupted culture from the baffled flasks wastransferred into 9 l of R20-2 medium with 2% glucose. The fermentor(Braun) was equipped with an impeller; internal heating coils andbaffles were removed. Fermentation conditions during the run were: 600rpm initially and increased to 1000 rpm after 44 hours to increasemixing of the culture medium which had became viscous. Temperature wascontrolled at 30° C. (-1° C.+3° C.). The pH was controlled at 5.0±0.1and oxygen concentration in the broth was maintained at about 30% of airsaturation. At 48 hours the contents of the fermentor were collected.The cellulose was allowed to settle and excess liquid was poured off.The remaining cellulose was blended, washed and filtered as describedabove. The cellulose was washed with deionized (DI) water, extracted in0.5M NaOH at 60° C. overnight. After the extraction the cellulose waswashed with DI until the pH of the wash water dropped below 6.0. Thepreparation was used for scanning electron microscopic examination asdescribed in Example VIII.

EXAMPLE VII

Preparation of Non-Reticulated Cellulose Product By Acetobacter Strain1306-8 in Static Culture

Strain 1306-8 is an Acetobacter isolate selected directly from a sampleof NRRL B42. Colonies of 1306-8 are characterized by white, highlyraised colonies on R20-2 plates. Strain 1306-8 produced gluconic acid. Asample of 1306-8 from a frozen stock (2.5 ml) was inoculated into 100 mlR20-2 medium and was cultured under static conditions at 30° C. in a 500ml wide mouth Erlenmeyer flask. The seed culture was incubated about 2-4days under these conditions until a visible pellicle formed. The entirecontents of the seed culture were then blended and used to inoculatesubsequent cultures.

Fermentation was carried out with wide-mouth Fernbach flasks containing1 liter of R20-2 medium with 0.025 ml of a 10 g/100 ml suspension ofBenlate fungicide (Dupont) in dimethyl formamide (DMF). Thisconcentration of DMF and Benlate was effective to prevent fungalcontamination without measurably affecting the growth of Acetobacterstrain 1306-8.

About 8-10 ml was used to inoculate 1 liter of R20-2 culture medium. Thecultures were grown in the Fernbach flasks for 10-14 days at 30° C.without agitation (i.e., standing cultures). At the time of harvest a0.3 to 1.2 cm thick pellicles had formed.

Pellicles were removed from Fernbach flasks, then blended and washedwith deionized water to remove media and part of the cells. Washing wasdone by filtration through a large buchner funnel using a filter screen(Spectramesh #146382) with mesh opening of 286 μM as a filter.

Extraction of blended and washed pellicle was carried out in 0.50 M NaOHat about 60° C. for about 14 hours. After mixing and incubating for thedesired period of time, the extraction mixture was filtered under theconditions described above. Washes with deionized H₂ O were continueduntil the pH of the wash water dropped below 6.0. The preparation wasused for scanning electron microscopic examination as described below inExample VIII.

EXAMPLE VIII

Microscopic Examination of Cellulose Products

The cellulose products obtained in Examples VI and VII above wereprepared for scanning electron microscopy (SEM). Specimens were freezedried and then sputtered under vacuum with a 60:40 gold:palladiumconductive film. Photomicrographs were taken using a Nanometricsscanning electron microscope operated at 16 Kv accelerating voltage.

FIGS. 2 and 4 are representative electron micrographs of the celluloseproduct obtained. FIGS. 2 and 4 show the cellulose product produced instatic cultures according to Example VII. The figures show that theproduct consists predominantly of piles of extended cellulose fibrilsthat appear to overlap and cross one another but do not appear tointerconnect. The filaments of cellulose produced under staticconditions ranged in width from about 0.05 to 0.2 microns with manystrands in the 0.05 to 0.1 micron range.

By contrast, FIGS. 3 and 5 show that the cellulose product produced inagitated culture in Example VI consists of a reticulum of fibrils thatare generally thicker in cross section--in a range between 0.1 to 0.2microns--than the cellulose grown in static culture. In addition thecellulose fibrils appear to form a network of predominantlyinterconnecting, rather than overlapping, fibrils.

EXAMPLE IX

Bacterial Cellulose products: Handsheets

This example shows some of the characteristics of the reticulatedcellulose product according to the invention. Handsheets were preparedfrom samples of Acetobacter-produced cellulose to an approximate basisweight of 60 g/m² according to the procedure described in TAPPI officialtest method T205 om-81. Cellulose was produced from strain 1306-3 understatic growth conditions in a flask culture using R20-medium with 2%glucose. Reticulated cellulose product was produced using strain 1306-11in a fermentor under agitation with an impeller at 600 rpm using thesame medium. The broth containing the cellulose was cold-stored prior toprocessing the cellulose. The cellulose was dispersed (1.2 g in 2 litersof water) in a British Disintegrator for 15,000 revolutions. Thesuspension was then poured into an automatic sheet mold containing a200-mesh wire screen and allowed to drain at least two hours. The moisthandsheet (15 cm in diameter) was removed from the sheet mold andinitially pressed gently between blotters to remove excess water. Thesheet was then placed in a TAPPI press between blotters for varyingtimes under a 50 psi (345 kPa) load to produce sheets of variousdensities. Final drying of the sheets was done by passing them through aNoble and Wood laboratory drum dryer.

Tensile strength of sheets was measured according to TAPPI method T494om-81 using an Instron Universal test instrument.

The reticulated cellulose product of this Example was found to offersubstantial resistance to densification when it was formed into ahandsheet. By use of different wet pressing loads, a series of sheetswas prepared having densities of about 300 to about 500 kg/m³. In spiteof the low densities, these paper-like sheets have very high tensilestrength. Typically, the tensile index for such sheets of the abovedensity ranges is between 100 and 150 Newton meters/gram. Comparablesheets formed from kraft pulp having densities below about 500 kg/m³have virtually no tensile strength.

Handsheets formed from cellulose produced under static cultureconditions do not exhibit the above-mentioned resistance todensification. Typically, such sheets from non-agitated cultures ofcellulose have densities from about 500 to about 750 kg/m³ depending onthe wet pressing load employed.

EXAMPLE X

Resistance to densification

This Example compares the resistance to densification of reticulatedcellulose produced under agitated culture conditions and non-reticulatedcellulose produced under static conditions in two Acetobacter strains.Reticulated cellulose was obtained using strain 1306-14 under agitatedgrowth conditions as described in Example VI. Non-reticulated cellulosewas obtained using strain 1306-3 in static flask cultures using R20-2medium -2% glucose under essentially the same static conditionsdescribed in Example VII.

Handsheets were prepared from the reticulated cellulose and staticallyproduced cellulose to an approximate weight basis of 60 g/m² asdescribed in Example IX except that various pressing loads were used toproduce the sheets. The density and other characteristics of sheetsproduced in this example from reticulated and non-reticulated celluloseare shown in Table 5.

                  TABLE 5    ______________________________________              Basis    Sample    Weight, Thickness, Density,                                       Pressing    No.       g/m.sup.2                      mm         kg/m.sup.3                                       Method*    ______________________________________    Non-reticulated              56.9    .106       538   A    cellulose    Reticulated              55.3    .219       252   A    cellulose    Non-reticulated              60.3    .080       753   B    cellulose    Reticulated              67.9    .157       433   B    cellulose    ______________________________________     *Method B includes higher pressing pressure than Method A.

EXAMPLE XI

Comparison of Cellulose Production By Various Acetobacter Strains

Six strains were tested. 1306-3 and 1306-11 were as described hereinabove. Two subcultures of Acetobacter aceti subsp. xylinum ATCCaccession number 23769, designated herein as 23769A and 23769B weretested. In addition, ATCC strain 31174 and National Collection ofIndustrial Bacteria strain 8132 (Aberdeen, U.K.), and strain 1499-1 werealso tested.

The growth medium for pre-seed culture, seed culture and productionstages was CSL medium with 4% (w/v) fructose and 5% (w/v) CSL.

Pre-seed cultures were grown in 100 ml of the above-described medium ina 750 ml Falcon #3028 tissue culture flask with 0.01% Dow Corningantifoam under static conditions at 30° C. for 24 to 48 hours. Theentire contents of the pre-seed culture was blended as describedpreviously and was used to make a 5% (v/v) inoculum of the seed culture.Pre-seeds were streaked on R20-2 plates to check for contamination. Allstrains had homogeneous colony morphology except strain 1499 which hadapproximately 50% large colonies.

Seed cultures were grown in 25 ml of the above-described medium inbaffled 125 ml flasks under shaking conditions in a reciprocal shaker at125 rpm at 30° C. for three days. A sample from each of the blendedseeds was streaked on R20-2 plates to check for contamination. Allstrains had homogeneous colony morphology except 1499-1 which hadapproximately 50% large colonies. The entire remaining contents of theseed culture was blended as described previously and was used to make a5% inoculum for the production stage.

Duplicate fermentation flasks of each strain were grown in 125 mlbaffled shake flasks on a reciprocal shaker at 125 rpm at 30° C.Duplicate flasks of each strain were harvested at days 1, 2, 3 and 4except for ATCC strain 23769B which was harvested on day 7 due to poorgrowth. Strain 1499-1 and strain ATCC 31174 both produced a watersoluble polysaccharide (WSP) under these conditions. No WSP was producedby strains 1306-3 or 1306-11.

Cellulose production was determined for each strain and is reported inTable 6. Values for cellulose production are in g/l.

                  TABLE 6    ______________________________________    Cellulose Production for Various Strains*    5% CSL, 4% Fructose    Strain      Day 1   Day 2     Day 3 Day 4    ______________________________________    1306-3      1.05    2.51      3.68  3.79    1306-11     1.25    3.09      4.45  5.55    ATCC 23769A 0.17    0.90      1.00  0.95    ATCC 23769B 0.13    0.54      0.62  N.D.**    1499-1      1.41    3.40      4.48  4.42    NCIB 8132   0.46    0.99      1.43  1.92    ATCC 31174  1.09    2.73      4.21  4.18    ______________________________________     *Values for cellulose production are in g/l     **N.D. = not determined

EXAMPLE XII

Cellulose Production of Acetobacter Strain 1306-11 in Fermentors

Pre-seed and seed cultures of 1306-11 were grown as in Example XI fortwo days except that the medium contained 4% w/v glucose and 5% w/v CSL.

Seed cultures were grown in two liter baffled flasks in the same mediumfor two days except that the culture volume was 400 ml.

Two 14 l Chemap fermentors were run with a 5% (v/v) inoculum in initial12 l volumes. During the 72 hour fermentor run, the cultures weremaintained at about 30° C. (±1° C.).

In fermentor #1 the initial glucose concentration was 32 g/l. Infermentor #2 the initial glucose and sucrose concentrations were 10 g/land 20 g/l, respectively. One hundred forty three g/l glucose wasadditionally added intermittently to fermentor #1 during fermentation.In fermentor #2, 50 g/l glucose and 72 g/l sucrose was addedintermittently during fermentation.

The initial 2% v/v CSL concentration was augmented by the addition of anamount equivalent to 2% by volume of the initial volume at 32 hours and59 hours.

The dissolved oxygen concentration was set at 30% air saturation. Itfell in fermentor #2 to zero at hour 69 for about two hours of thefermentor run. Agitation was maintained initially at 600 rpm. Asviscosity increased with increasing cellulose concentration, theimpeller speed was increased to 1200 rpm. The concentrations ofcellulose, gluconic acid, and 2-keto-gluconic acid are shown forfermentor 1 in Table 7. The concentration of cellulose is shown forfermentor 2 in Table 8. The maximum cellulose concentration reached inthe glucose only fermentor #1 was 12.7 g/l. The maximum celluloseconcentration reached in the glucose/sucrose fermentor #2 was 18.3 g/l.Both maxima were reached at 71.6 hours into the fermentation. Thevolumetric productivities at this time in fermentor #1 and #2 were 0.18and 0.26 g/l/hr., respectively.

                  TABLE 7    ______________________________________    Fermentor #1: 2% (v/v) CSL + 3.2% (w/v) Glucose (Initial)            Cellulose  Gluconic Acid                                  2-Keto-Gluconic    Hours   g/l        g/l        Acid g/l    ______________________________________    0.5     0.1        0.09       0.5    8.0     0.3        0.21       --    18.0    1.6        0.38       1.0    22.5    2.8        0.53       2.0    28.2    4.6        0.91       3.4    32.8    5.7        1.29       5.0    42.2    7.9        2.71       --    45.7    9.3        4.28       14.6    51.5    --         5.92       --    55.8    11.2       --         --    57.3    11.1       7.27       25.5    69.0    11.3       9.01       35.0    71.6    12.7       9.29       36.9    ______________________________________

                  TABLE 8    ______________________________________    Fermentor #2: 2% (v/v) CSL + 1% (w/v) Glucose +    2% (w/v) Sucrose (Initial)                 Cellulose           Hours g/l    ______________________________________           0.5   0.2           8.0   0.4           18.0  1.5           22.5  2.7           28.3  4.4           32.9  6.1           42.2           45.8  10.5           51.5  13.0           55.8           57.3  14.5           69.0  17.0           71.6  18.3    ______________________________________

EXAMPLE XIII

Cellulose Production By Acetobacter Strain 1306-21 in Fermentors

The fermentation described in Example XII was repeated using strain1306-21 in place of 1306-11 and with the following changes:

1. The preseed and seed were prepared in CSL medium with 2% glucose, 2%corn steep liquor.

2. The agitation rate did not exceed 900 rpm.

3. The initial glucose concentration was 20 g/l, and 109 g/l more wereadded during the run.

4. Initial CSL concentration was 2% v/v and 2% v/v more were added after27.8 hours in the run.

The concentrations of cellulose, gluconic acid, 2-keto-gluconic acid and5-keto-gluconic acid observed in this run are reported in Table 9.

                  TABLE 9    ______________________________________    14-L Chemap Fermentation: Strain 1306-21                                 2-Keto-                                        5-Keto-             Cellulose                      Gluconic   gluconic                                        gluconic    Hours    g/l      Acid g/l   Acid g/l                                        Acid g/l    ______________________________________    0.5      0.18     N.D.       N.D.   N.D.    13.3     0.60     N.D.       N.D.   N.D.    20.8     1.75     N.D.       N.D.   N.D.    27.8     2.55     N.D.       N.D.   N.D.    37.1     5.67     N.D.       N.D.   N.D.    44.8     8.74     N.D.       N.D.   N.D.    52.2     10.90    N.D.       N.D.   N.D.    59.0     12.76    0.80       N.D.   N.D.    ______________________________________     N.D. ≦ 0.5 q/l

Comparing the results of Table 9 with those of Table 7, it appears thatstrain 1306-21 is equal to or better than strain 1306-11 as regardscellulose production while producing much less acid. Lower acidproduction should, in theory, permit strain 1306-21 to be grown tocomparable concentrations with less base addition.

EXAMPLE XIV

Effects of Agitation on Cellulose Properties

Two sets of tests were carried out to assess the effect of agitation onthe following four properties:

1. Handsheet formation (TAPPI test of Example IX): the ability ofpurified, treated and resuspended cellulose fibers from the fermentationto form an integral sheet on a 150 mesh screen. The results are judgedaccording to the percent of the resuspended fibers retained on thescreen (% cellulose retention) and to a qualitative assessment of theintegrity of the sheet formed.

2. Settling rate: the rate at which a diluted sample of cellulose fromthe fermentor settles in a graduated cylinder. The decreasing height ofthe sediment/supernatant interface of the settling suspension ofcellulose is plotted versus time. The instantaneous settling rate can bedetermined from the slope of the plot at a given time. This propertydepends on the cellulose particle size and density.

3. Suspension viscosity: the viscosity of suspended cellulose fermentorbroth measured by a Thomas-Sormer viscometer calibrated with glycerolsolutions. This property depends on the morphology of the celluloseparticles.

4. Particle size and morphology: This property is determined fromphotomicrographs of cellulose from the fermentor.

The first set of tests consisted of four fermentor runs, usingAcetobacter strain 1306-11, during which samples were withdrawn as afunction of time from 14 liter Chemap fermentors operated at varyingrates of maximum agitation. These four fermentor runs were typicalfermentations with growing cultures, but extended past normalfermentation times to allow evaluation of the effect of agitation overprolonged time. Samples from these runs were analyzed for handsheetformation ability.

The second set of experiments, also using Acetobacter strain 1306-11,consisted of agitating old, non-growing cellulose cultures, harvestedfrom a 250 liter fermentor run, in four 14 liter Chemap fermentors. Theagitation rate was constant in each fermentor, but was varied amongfermentors. Samples were taken with respect to time form each fermentor.The cellulose con centration in this set of experiments was uniform inall the fermentors, but was approximately half the concentratration ofthe final concentrations in the first set of experiments. In this secondset of experiments, nitrogen was sparged at about the average gassingrate (air and oxygen) of the first set of experiments. Samples from thesecond set of experiments were analyzed for handsheet formation,settling rate, viscosity, and particle size and morphology.

Handsheet results from the first and second sets of experiments aresummarized in Tables 10 and 11, respectively.

                  TABLE 10    ______________________________________    Agitation             Time at This   Sheet    % Cellulose    Rate (rpm)             Agitation Rate (hr)                            Formation                                     Retention    ______________________________________    700      18             Good*    100             38             Good*    92             81             Poor*    64    750      16             Good*    94             37             Good*    101             81             Poor*    55    800      16             Good*    109             38             Poor*    90             81             Poor*    84    1,000    0              Good     102             23             Poor     38             50             None     0             64             None     0    ______________________________________     *These sheets had a target weight of 0.65 to 0.75 g cellulose instead of     the usual 1.2 g, which was the target weight for the 1000 rpm run.

                  TABLE 11    ______________________________________    Agitation           Time at This    Sheet     % Cellulose    Rate (rpm)           Agitation Rate (hr)                           Formation*                                     Retention    ______________________________________    600    16              Good      96           40              Good      99           63              Good      89           86              Good      88    700    16              Good      92           40              Good      89           63              Good      88           86              Good      89    800    16              Good      90           40              Good      86           63              Good      100           86              Good      92    1,000  16              Good      81           27              Good      96           50              Poor      73           73              Poor      78    ______________________________________     *All of these sheets had a target weight of 1.2 g.

Table 10 shows that handsheet formation may be adversely affected byboth increased agitation rate and increased time of agitation.

The results in Table 11 in which more dilute and non-growing cellulosecultures were used showed handsheet formation to be not as sensitive tothe agitation rate and time as shown in Table 10. Good handsheets couldbe made up to 86 hours at 800 rpm or 27 hours at 1000 rpm agitation. Thebetter results in Table 11 may be due to lower cellulose concentrationin the fermentor or higher target sheet weights. In addition the pelletsmay have been more resilient to start with due to different fermentationconditions.

Other test results do, however, show cellulose properties being affectedby agitation rate and time during the second set of experiments. Forinstance, the viscosity analyses indicate increasing viscosity of thecellulose suspension in the fermentor with agitation time at 800 and1000 rpm. The increased viscosity may reflect the change in themorphology of the cellulose from dense pellets to a less densely packedfiberlike form. Such changes were observed in the photomicrographs usedto evaluate particle size and morphology.

The settling rate studies showed that cellulose agitated at higher ratesor for longer times settled more slowly. These results were consistentwith the photomicrographs which showed that with increased agitationrate and time the cellulose pellets appear fragmented into smallerparticles as determined by light microscopy. The results from analysesof the settling rates, viscosities, particle size and form, andhandsheet formation, all appear generally consistent and to some extent,correlate with each other.

The mechanism of change of the cellulose properties with agitation rateand time is not understood completely at this time. It is known thatincreased agitation rates increase the shear stress on the celluloseparticles. However, other forces may also be contributing to thechanging cellulose properties; examples include turbulent eddy stressesand flocculating pressures. Furthermore, the above forces and stresses,as well as others, are also exerted during recovery and purification ofthe cellulose. Accordingly, caution must be exercised during all stagesof cellulose processing to minimize damage to the cellulose.

EXAMPLE XV

The Effect of Citrate, Nitrilotriacetic Acid, and Ferric Iron onAcetobacter Growth

Acetobacter strain 1306-21 was adapted to growth in the absence ofvitamins in the medium. The adaptation was made by subculturing it 20times in AMM (R-70) w/out vitamins. The basal medium used in theexperiment was AMM w/out vitamins +3% (w/v) glucose, 25 mM 3,5-dimethylglutaric acid (DMG), 1 μM CoCl2, and 1 μM NiCl2.

The preseed for the experiment was grown in basal medium in standingflasks for 48 hr. The seed was grown in basal medium for 72 hr (30° C.,125 rpm). All test flasks received a 6% (v/v) inoculum. The flasks wereincubated at 30° C., 125 rpm, and harvested after three days. Biomassanalysis (cellulose+cells) was used to follow cell growth. Cells pluscellulose were centrifuged, washed with deionized water and dried in avacuum oven at 80° C. to a constant weight.

The results are shown in Table 12. Although there are some majordifferences between duplicate flasks, the results show that all of thetest systems had significantly more growth than did the basal mediumcontrol. The addition of ferric iron salt stimulated growth as well asthe addition of citrate or nitrilotriacetic acid (NTA). Both citrate andNTA are known to be strong chelators of ferrous and ferric ions.

                  TABLE 12    ______________________________________    The Effect of Citrate, Nitrilotriacetic Acid,    and Ferric Iron on Acetobacter Growth                           Average                    Biomass                           Biomass                    (g/L)  (g/L)    ______________________________________    Basal Alone       0.820    0.774                      0.728    Basal + 5 mM Citrate                      2.404    1.912                      1.420    Basal + 10 μM FeCl.sub.3                      0.952    1.280                      1.608    Basal + 1 mM Citrate +                      2.308    1.888    10 μM FeCl.sub.3                      1.468    Basal + 1 mM      1.756    1.616    Nitrilotriacetic Acid                      1.476    Basal + 0.2% Peptone                      1.640    1.582                      1.524    Basal + 0.3% TYE  2.464    2.416                      2.368    ______________________________________

EXAMPLE XVI

Evaluation of a New Medium (R70-2)

The previous example (XV) suggested that iron is limiting in AMM (R70)medium. The precipitate that has been observed in this medium may beiron phosphate. Addition of iron chelators like citric acid ornitrilotriacetic acid prevents the precipitation of iron and enhancesgrowth. On the basis of these findings a new medium was formulated with4 mM (Na)citrate. The purpose of this study was to evaluate Acetobactergrowth in the new medium (R70-2) compared to the old medium (R70).

The ingredients of AMM (R70) and R70-2 are set forth above. The newmedium (R70-2) had the following changes relative to R70:

1. 0.010 mM FeCl₃ was used instead of 0.013 mM FeSO₄ ;

2. 4 mM citrate was present;

3. 0.001 mM CoCl₂ was present;

4. 0.001 mM NiCl₂ was present;

5. CuSO₄ was increased from 0.0002 mM to 0.001 mM;

6. ZnSO₄ was decreased from 0.006 mM to 0.005 mM; and

7. MnSO₄ was decreased from 0.006 mM to 0.005 mM

The (NH₄)₂ SO₄, KH₂ PO₄, MgSO₄ and citrate were prepared at 1×concentration and steam sterilized. The trace metal solution (includingiron) and the vitamin mix were prepared at 100× concentration, filtersterilized, and added aseptically.

The Acetobacter strain used was 1306-21. The preseed for this experimentwas grown for 48 hr in R70-2 containing 2% (w/v) glucose,. 0.2% (w/v)technical grade extract, Amberex 1003 (Universal Food, WI), (TYE) and 25mM DMG. The seed was grown for 72 hours in R70-2 with 2% glucose, 0.1%(w/v) TYE and 25 mM DMG. The test flasks had 2% (w/v) glucose and 25 mMDMG. The inoculum was 5% (v/v), and the flasks were incubated at 30° C.and 125 rpm. Biomass measurements were used to follow cell growth.

The results are shown in FIG. 6. At both low and moderate yeast extractconcentrations, the new medium supported significantly higher levels ofgrowth than R70 medium. The 0.3% TYE R70-2 flasks were glucose-limitedon day 4 (less than 0.1% glucose was present). Higher levels of biomassmay be obtainable if the glucose is kept in excess. All of the othersystems contained excess glucose.

The R70-2 plus 0.03% TYE did not reach stationary phase even after 4days of incubation. The pH of the day 4 flasks was 3.8 to 4.0.Presumably, the pH of the medium would start to inhibit growth if theflasks were left on the shaker for a longer period of time.

EXAMPLE XVII

Cellulose Production with Technical Grade Yeast Extract (TYE) as aComplex Nitrogen Source

This Example compares cellulose production by Acetobacter on 0.4% TYEversus 0.4% TYE plus 0.1% CSL.

The basal fermentor medium was R70-2 medium with the followingmodifications:

1. The (NH₄)₂ SO₄ was decreased to 12.5 mM;

2. Initial glucose concentration was 2% (w/v);

3. 0.4% (w/v) TYE (Amberex 1003, Universal Foods) was added to bothfermentors;

4. 0.1% (v/v) CSL was added to one of the fermentors before inoculation;and

5. Initial concentrations of phosphate, magnesium, and calcium weredoubled.

Acetobacter strain 1306-21 was used for both fermentors. The seed wasgrown according to standard procedures in R70-2 with 3% glucose, 25 mMDMG, and 0.5% TYE. Both seed stages were grown for 2 days beforetransfer.

NH₄ OH (4N) was used to titrate acid production during the fermentor runas well as to supply additional inorganic nitrogen. Glucose addition waslinked to the base addition to maintain a more even glucoseconcentration. The fermentors used were 14-L Chemap (Switzerland)fermentors.

Tables 13 and 14 show the kinetics of cellulose production with 0.4% TYEand 0.1% CSL, respectively. The results demonstrate that TYE can be aneffective complex nitrogen source and that addition of CSL enhancescellulose production.

                  TABLE 13    ______________________________________    Cellulose Production with 0.4% TYE            Time Cellulose            (hr) (g/l)    ______________________________________            0.81 0.19            19.72                 0.71            28.32                 1.19            34.90                 1.62            43.92                 2.19    ______________________________________

                  TABLE 14    ______________________________________    Cellulose Production with 0.4% TYE    and 0.1% CSL            Time Cellulose            (hr) (g/l)    ______________________________________            0.83 0.19            19.69                 1.59            24.64                 2.87            29.60                 4.68            34.87                 6.16            43.92                 6.97    ______________________________________

EXAMPLE XVIII

Cellulose Production with Sheftone F as a Complex Nitrogen Source

Shake flask experiments identified Sheftone F (Sheffield, Norwich, N.Y.)as an effective complex nitrogen source-for cellulose production. ThisExample compares cellulose production using 1% Sheftone F and 0.2% TYEversus 1% Sheftone F plus 0.2% CSL as a complex nitrogen source infermentors.

The basal fermentor medium was R70-2 with the following modifications:

1. The (NH₄)₂ SO₄ was decreased to 12.5 mM;

2. Initial glucose concentration was 2% (w/v);

3. 1.0% (w/v) Sheftone F was added to both fermentors;

4. 0.2% (w/v) TYE was added to one fermentor;

5. 0.2% (v/v) CSL was added to the second fermentor; and

6. Initial concentrations of phosphate, magnesium, and calcium weredoubled.

Acetobacter strain 1306-21 was used for both fermentors. The strain wasgrown as described in Example XVII in R70-2 with 3% (w/v) glucose, 25 mMDMG, and 0.5% (w/v) TYE. Both seed stages were grown for 2 days beforetransfer.

NH₄ OH (4N) was used to titrate acid production during the fermentor runas well as to supply additional inorganic nitrogen. Glucose addition waslinked to the base addition to maintain a more even glucoseconcentration. The fermentors used were 14-L Chemap fermentors.

The kinetics of cellulose production on 1% Sheftone F supplemented with0.2% TYE or CSL are presented in Tables 15 and 16, respectively. Theseresults demonstrate that Sheftone F is an effective complex nitrogensource for cellulose production.

                  TABLE 15    ______________________________________    Cellulose Production with 1%    Sheftone and 0.2% TYE            Time Cellulose            (hr) (g/l)    ______________________________________            0.73 0.07            17.47                 0.64            21.56                 1.12            25.97                 1.81            30.66                 2.85            40.93                 6.33            48.11                 7.68            55.35                 10.53            66.15                 9.97    ______________________________________

                  TABLE 16    ______________________________________    Cellulose Production with 1%    Sheftone and 0.2% CSL            Time Cellulose            (hr) (g/l)    ______________________________________            0.76 0.09            17.58                 0.66            21.48                 1.14            26.18                 1.89            30.71                 2.91            41.27                 6.69            48.09                 8.26            55.40                 10.49            66.30                 11.16    ______________________________________

EXAMPLE XIX

C-13 NMR Analysis of Cellulose Products

The microstructure of the bacterial cellulose produced under agitatedconditions (as described in Table 19) was examined and compared to thatof bacterial cellulose produced under non-agitated or static cultureconditions. NMR spectrometry was performed essentially as described byVanderhart et al., in Macromolecules 17:1465-1472 (1984), incorporatedby reference herein, except with certain modifications. Thus, NMRspectrometry was performed using an S-100 NMR spectrometer (GeneralElectric, Fremont, Calif.) operating at 2.34 T, which corresponds tofrequencies of 100.2 MHz for protons and 25.2 MHz for C-13.Cross-polarization times were typically 1.0 to 2.0 ms.

The respective radiofrequency amplitudes were set for a Hartmann-Hahnmatch at a rotating-frame precision frequency of 43 kHz, and notmismatched by the sample spinning frequency. The magic-angle spinningrate was 2800-3100 rps. Chemical shift referencing was accomplished bysetting operating frequencies daily so that the methyl carbon resonanceof hexamethylbenzene appeared at 17.80 ppm. The rotor material was boronnitride or phase-stabilized zirconia.

The NMR results for the samples tested are set out in Tables 17 and 18below and consisted of cellulose produced by five (5) non-agitatedAcetobacter cultures and from eleven (11) Acetobacter cultures that werecultured in fermentors. The strains used and culture conditionscorresponding to the sample numbers in Tables 17 and 18 are set forth inTable 19.

Tables 17 and 18 present the resulting morphology distribution obtainedby NMR analysis of the samples indicated therein from strains culturedstatically and in agitated culture. These results show that there areimportant differences between cellulose produced from fermentor samples(agitated) compared to that from static culture. The lowercrystallinities (cellulose I) of agitated samples are supported by thesubstantial changes in the amounts of the I.sub.α and I.sub.β spectrumpeaks and the peaks due to amorphous cellulose. Especially noteworthy isthe consistent presence of cellulose II in all fermentor samples and itsabsence (except for sample A-008 corresponding to Acetobacter strain1307) in static culture samples. In addition, in two agitated samples(samples A-070 and A-071 corresponding to Acetobacter strain 1306-11),subject to long fermentation times and thus; high shear stress, asignificant portion of the cellulose is cellulose II, suggesting arelationship between cellulose II content and the amount of agitation inculture. There was also a consistent difference between the I.sub.αcontent in static as compared to agitated cultures. In addition, ahigher level of residual signal occurs at 90 ppm in cellulose fromagitated culture. As shown in Tables 17 and 18 the method providesexcellent agreement between the two determinations of amorphouscellulose content.

Although not wishing to be limited by any particular explanation, thedifferences observed herein using NMR analysis may reflect the manner inwhich individual cellulose molecules are packed together and toconformational differences between different molecular chains.

                  TABLE 17    ______________________________________    NMR Studies Of Bacterial Cellulose Morphology    MOLE % (AVG. DEVIATION) OF CRYSTALLITE                           AMORPHOUS    SAMPLE I.sub.α                   I.sub.β                           II   RESIDUAL*                                         SUB   C-4    ______________________________________    Static Culture Samples    A-007  40 (1)  26 (2)  --   4 (1)    29 (<1)                                               29    A-008  34 (<1) 21 (<1) 7 (2)                                6 (1)    32 (<1)                                               32 (1)    A-009  42      25      --   3        30    29    A-010  42 (1)  28 (1)  --   2        28 (<1)                                               26 (<1)    A-012-3           38 (<1) 27 (<1) --   4        31 (<1)                                               31 (<1)    Subspectrum Sources    A-012-6           42      30      --   --       28    27    (also    static)    Whatman           16      51      --   --       33    33    CF-1    (from    Cotton)    ______________________________________     *Residual Component at 90 PPM     -- Not detectable

                  TABLE 18    ______________________________________    NMR Studies Of Bacterial Cellulose Morphology    Fermenter (Agitated) Samples    MOLE % (AVG. DEVIATION) OF CRYSTALLITE                           AMORPHOUS    SAMPLE I.sub.α                   I.sub.β                           II   RESIDUAL*                                         SUB   C-4    ______________________________________    A-070  19      15      24   7        35    38    A-071  19      18      25   4        34    34    A-072  23 (1)  24 (1)  6 (2)                                10 (1)   37 (<1)                                               38 (1)    A-075  22 (<1) 19 (<1) 8 (1)                                11 (2)   40 (1)                                               38    A-076  22      23      15   4        36    37    A-085  29      26      8    4        32    31    A-091  22      19      12   11       36    35    A-092  22      17      12   14       35    37    A-095  25      21      6    14       34    35    A-125  23      16      13   12       36    36    A-126  30      23      6    8        34    32    ______________________________________     *Residual Component at 90 PPM

                  TABLE 19    ______________________________________    Strains And Culture Conditions For NMR    Studies Of Bacterial Cellulose Morphology    Sample          Strain   Fermentation  Post Fermentation    Number          Source   Conditions    Treatment    ______________________________________    A-007 1306-3   f,st,G/R20    0.8 M NaOH, 32° C., 12 h    A-008 1307 sm2.sup.1                   "             0.5 M NaOH, 60° C., 1 h    A-009 1499-1   "             0.5 M NaOH, 50° C., 12 h    A-010 1306-3   "             0.5 M NaOH, 60° C., 16 h    A-012 1306-8   "             0.5 M NaOH, 60° C., 1 h    A-070 1306-11  F/600-1000 rpm                   G + CSL       0.5 M NaOH, 60° C., 2 h    A-071 1306-11  F/600-1200 rpm                                 0.5 M NaOH, 60° C., 2 h                   G + S + CSL    A-075 1306-11  F/600-1100 rpm                                 0.5 M NaOH, 60° C., 2 h                   G + S + CSL    A-076 1306-11  F/600-950 rpm 0.5 M NaOH, 60° C., 2 h                   F + CSL    A-085 1306-11  250L/100-185 rpm                                 0.1 N NaOH, 60° C., 2 h                   G + CSL    A-091 1306-11  F/600-1000 rmp                                 0.1 N NaOH, 65° C., 2 h                   G + CSL    A-092 1306-11  F/600-1000 rpm                                 0.1 N NaOH, 65° C., 2 h                   G + CSL    A-095 1306-11  750L/100-150 rpm                                 0.1 N NaOH, 65° C., 2 h                   G + CSL       followed by light column                                 washing and thorough                                 washing in filter press    A-125 1306-11  250L/100-175 rpm                                 0.1 N NaOH, 65° C., 2 h                   G + CSL    A-126 1306-11  6000L/40-60 rpm                                 0.1 N NaOH, 65° C., 3.5 h                   G + CSL       large-scale preparation    ______________________________________     Abbreviations: f = flask; F = fermentor; st = static; G = glucose; S =     sucrose; F = fructose; CSL = corn steep liquor; 250L = 250L fermentor.     .sup.1 Strain 1307 sm2 was a variant of Acetobacter strain 1307

It will be appreciated from the foregoing that bacterial cellulose maybe produced at high efficiency under agitated conditions for sustainedperiods of time. Heretofore the sustained production of bacterialcellulose at high productivity has been fraught with difficulties andextremely low productivities. The invention disclosed herein and claimedbelow clearly represents a major advance in fermentative production ofbacterial cellulose.

Deposits

Samples of strains 1306-3, 1306-11, 1306-21, 1306-8 and 1306-14 weredeposited under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure and Regulations thereunder at the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. USA 20832. Depositdates and accession numbers are given below:

    ______________________________________    Strain   Accession No.   Deposit Date    ______________________________________    1306-3   53264           September 13, 1985    1306-11  53263           September 13, 1985    1306-21  53524           July 25, 1986    1306-8   53749           March 1, 1988    1306-14  53750           March 1, 1988    ______________________________________

Said deposits were made pursuant to a contract between the ATCC and theassignee of this patent application. The contract with the ATCC providesfor permanent availability of said strains and progeny thereof to thepublic upon issuance of a U.S. patent related to this applicationdescribing and identifying the deposit or upon the publication or layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and for the availability of these strains and the progenythereof to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 USC §122 and theCommissioner's rules pursuant thereto (including 37 CFR §1.14 withparticular reference to 886 OG 638). The assignee of the presentapplication has agreed that if the strains on deposit should die or belost or destroyed when cultivated under suitable conditions, it will bepromptly replaced upon notification with a viable culture of the samestrain.

The deposits under the terms of the Budapest Treaty assures that saidcultures deposited will be maintained in a viable and uncontaminatedcondition for a period of at least five years after the most recentrequest for the furnishing of a sample of the deposited microorganismwas received by the ATCC and, in any case, for a period of at least 30years after the date of the deposit.

Availability of the deposited strains is not to be construed as alicense to practice the invention in contravention of the rights grantedunder the authority of any government in accordance with its patentlaws.

Also, the present invention is not to be considered limited in scope bythe strains deposited, since the deposited embodiments are intended onlyto be illustrative of particular aspects of the invention. Anymicroorganism strains which are functionally equivalent to thosedeposited are considered to be within the scope of this invention.Further, various modifications of the invention in addition to thoseshown and described herein apparent to those skilled in the art from thepreceding description are considered to fall within the scope of theappended claims.

With the information contained herein, various departures from theprecise description of the invention will be readily apparent to thoseskilled in the art to which the invention pertains without departingfrom the spirit of the invention claimed below.

What is claimed is:
 1. A method of producing a reticulated cellulosecomprising type II crystalline cellulose as measured by nuclear magneticresonance, said method comprising the steps of:a) culturing underagitated culture conditions sufficient to prevent pellicle formation amicroorganism of the genus Acetobacter, or a mutant thereof, capable ofproducing said reticulated cellulose in said agitated cultureconditions, in a liquid medium suitable for cellulose production for aperiod of time sufficient to produce said reticulated cellulose, whereinsaid microorganism is stable against conversion from cellulose producingforms to cellulose non-producing forms in said agitated cultureconditions; and b) recovering said reticulated cellulose comprising saidtype II cystalline cellulose.
 2. The method of claim 1, wherein thereticulated cellulose is produced at an average volumetric productivityof at least 0.1 grams/liter/hr.
 3. The method of claim 1 wherein saidmicroorganism is selected from the group consisting of microorganismshaving all the identifying characteristics of ATCC Nos. 53264, 53749,53263, 53750 and 53524, and progeny thereof.
 4. The method of claim 1,wherein said microorganism produces reduced amounts of gluconic acid andketogluconic acids when grown in glucose-containing medium as comparedwith the production of said acids by Acetobacter strain ATCC No. 53264.5. The method of claim 4 wherein said microorganism is capable ofconverting less than 0.5% of glucose utilized by the microorganism inthe culture to gluconic acid and ketogluconic acids.
 6. The method ofclaim 1, wherein said microorganism is capable of converting fromcellulose producing forms to cellulose non-producing forms in saidagitated culture conditions at a frequency of less than 5×10⁻³ over thecourse of 42 to 45 generations as determined by observing colonymorphology of cells from the agitated culture on agar plates containinga carbon source.
 7. The method of claim 1 wherein said culturing iscarried out in a fermentation system selected from the group consistingof batch, fed batch, repeated batch and continuous fermentation.
 8. Themethod of claim 1, wherein said culturing is carried out in batchculture and the concentration of said reticulated cellulose produced isat least 10 g/l.
 9. The method of claim 1 wherein the recoveredreticulated cellulose comprising said type II crystalline cellulose hasa reticulated structure having strands of cellulose that interconnectforming a grid-like pattern extending in three dimensions to give afenestrated appearance as determined by scanning electron microscopy.10. The method of claim 1 wherein the liquid medium suitable for saidcellulose production comprises an assimilable carbon source, anassimilable nitrogen source, inorganic salts and vitamins suitable forgrowth of the cellulose producing microorganism.
 11. The method of claim10 wherein said assimilable nitrogen source is selected from the groupconsisting of casein hydrolysate, yeast extract, malt extract, peptone,ammonium salts, and corn steep liquor.
 12. The method of claim 10wherein the assimilable carbon source is selected from the groupconsisting of monosaccharides, disaccharides and mixtures thereof. 13.The method of claim 10 wherein the assimilable carbon source is a planthydrolysate selected from the group consisting of wood hydrolysates,straw hydrolysates, corn stalk hydrolysates and sorghum hydrolysates.14. The method of claim 1 wherein said liquid medium contains achelating agent.
 15. The method of claim 14 wherein said chelating agentis selected from the group consisting of citric acid salt andnitrilotriacetic acid salt.
 16. A method of producing a reticulatedcellulose comprising type II crystalline cellulose as measured bynuclear magnetic resonance, said method comprising the steps of:a)culturing under agitated culture conditions sufficient to preventpellicle formation, a microorganism of the genus Acetobacter, or amutant thereof, capable of producing cellulose in said agitated cultureconditions, in a liquid medium suitable for cellulose production at anaverage volumetric productivity of at least 0.1 g/L/hr, saidmicroorganism having a frequency of change in said agitated cultureconditions from cellulose producing forms to cellulose non-producingforms of less than 0.5% over the course of 42-45 generations asdetermined by observing colony morphologies of cells from the culture onagar plates containing a carbon source; and b) recovering saidreticulated cellulose comprising said type II cystalline cellulose. 17.The method of claim 16 wherein said Acetobacter microorganism is capableof converting less than 0.5% of glucose utilized by the microorganism inthe culture to gluconic acid and ketogluconic acids.
 18. The method ofclaim 16, wherein said culturing is carried out in a fermentation systemselected from the group consisting of batch, fed batch, repeated batchand continuous fermentation.
 19. The method of claim 16, wherein saidmicroorganism is Acetobacter strain ATCC No. 53264, 53749, 53263, 53750or 53524, or progeny thereof.
 20. The method of claim 16, wherein saidmicroorganism produces reduced amounts of gluconic acid and ketogluconicacid when grown in glucose-containing medium as compared with theproduction of said acids by Acetobacter strain ATCC No. 53264.